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1 Mechanisms of Infectious Disease • Fall 2006 Jonathan Dworkin, PhD Department of Microbiology [email protected] Genetic Basis of Variation in Bacteria I. Organization of genetic material in bacteria a. chromosomes b. plasmids II. Genetic variation: Source a. point mutation b. DNA rearrangements III. Genetic variation: Transmission a. transformation b. transduction c. conjugation IV. Genetic variation: Implications for pathogenesis Genetic Basis of Variation in Bacteria I. Organization of genetic material in bacteria a. chromosomes b. plasmids II. Genetic variation: Source a. point mutation b. DNA rearrangements III. Genetic variation: Transmission a. transformation b. transduction c. conjugation IV. Genetic variation: Implications for pathogenesis Genetic Basis of Variation in Bacteria I. Organization of genetic material in bacteria a. chromosomes b. plasmids II. Genetic variation: Source a. point mutations b. DNA rearrangements III. Genetic variation: Transmission a. transformation b. transduction c. conjugation IV. Genetic variation: Implications for pathogenesis Genetic Basis of Variation in Bacteria I. Organization of genetic material in bacteria a. chromosomes b. plasmids II. Genetic variation: Source a. point mutations b. DNA rearrangements III. Genetic variation: Transmission a. transformation b. transduction c. conjugation IV. Genetic variation: Implications for pathogenesis Genetic Basis of Variation in Bacteria I. Organization of genetic material in bacteria a. chromosomes b. plasmids II. Genetic variation: Source a. point mutations b. DNA rearrangements III. Genetic variation: Transmission a. transformation b. transduction c. conjugation IV. Genetic variation: Implications for pathogenesis

Genetic Basis of Variation in Bacteria - Columbia University · Genetic Basis of Variation in Bacteria I. Organization of genetic material in bacteria a. chromosomes ... Genetic basis

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Mechanisms of Infectious Disease • Fall 2006

Jonathan Dworkin, PhD

Department of Microbiology

[email protected]

Genetic Basis of Variation in Bacteria

I. Organization of genetic material in bacteriaa. chromosomesb. plasmids

II. Genetic variation: Sourcea. point mutationb. DNA rearrangements

III. Genetic variation: Transmission a. transformationb. transductionc. conjugation

IV. Genetic variation: Implications for pathogenesis

Genetic Basis of Variation in Bacteria

I. Organization of genetic material in bacteriaa. chromosomesb. plasmids

II. Genetic variation: Sourcea. point mutationb. DNA rearrangements

III. Genetic variation: Transmission a. transformationb. transductionc. conjugation

IV. Genetic variation: Implications for pathogenesis

Genetic Basis of Variation in Bacteria

I. Organization of genetic material in bacteriaa. chromosomesb. plasmids

II. Genetic variation: Sourcea. point mutationsb. DNA rearrangements

III. Genetic variation: Transmission a. transformationb. transductionc. conjugation

IV. Genetic variation: Implications for pathogenesis

Genetic Basis of Variation in Bacteria

I. Organization of genetic material in bacteriaa. chromosomesb. plasmids

II. Genetic variation: Sourcea. point mutationsb. DNA rearrangements

III. Genetic variation: Transmission a. transformationb. transductionc. conjugation

IV. Genetic variation: Implications for pathogenesis

Genetic Basis of Variation in Bacteria

I. Organization of genetic material in bacteriaa. chromosomesb. plasmids

II. Genetic variation: Sourcea. point mutationsb. DNA rearrangements

III. Genetic variation: Transmission a. transformationb. transductionc. conjugation

IV. Genetic variation: Implications for pathogenesis

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Genetic basis of variation: Griffiths (1928)

Genetic basis of variation: Avery et.al (1944)

Genetic basis of variation: Hershey and Chase (1952)

Organization of genetic material in bacteria: chromosomes

• Most bacteria contain a single chromosome (+ extrachromosomal elements)

• Some bacteria have been found also to contain 2-3 replicons which can be considered either megaplasmids or minichromosomes e.g. 3.0 Mb and 0.9 Mb replicons in Rhodobacter sphaeroides

• A few bacterial genera contain >1 chromosome e.g. 2.1 Mb and 1.2 Mb chromosomes in Brucella

• Some bacteria harbour large replicons essential for survival in a specific ecological niche but not under laboratory conditions e.g. 1.4 Mb and 1.7 Mb replicons inRhizobium meliloti are required for plant symbiosis

Organization of genetic material in bacteria: chromosomes

Organization of genetic material in bacteria: chromosomes

Copyright ©2004 by the National Academy of Sciences

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Organization of genetic material in bacteria: plasmids

Replication

Maintenance

Antibiotic-resistance

Transfer

Virulence

Organization of genetic material in bacteria: plasmids

Plasmid

Chromosome

• Extrachromosomal• Circular or linear• 2 kb to hundreds of kb in size• Non-essential• May carry ‘supplemental’ genetic information or may be cryptic• Employ host functions for most of DNA metabolism

http://www.biochem.wisc.edu/inman/empics/dna-prot.htm

Organization of genetic material in bacteria: plasmids

prgN uvrAsin

orf5txeaxeorf8

orf9

tnpA (IS6)ΔrepAΔaphA

orfY

sat4

aadE

orf19 orfXtnp (IS1182)ermB

tnpA (IS6)ΔrepM

tnp (IS16)

cat

mobΔrlx

tnpA (IS6)

repA

orf30

sojtnp (IS16)

orf2

orf10

orf15

pRUM24,873-bp

Transposon genes

Replication and maintenance

Antibiotic resistance

Other

Organization of genetic material in bacteria: plasmidsExamples of naturally-occuring plasmids and relevant features

Plasmid Host Plasmid size (kb) Relevant feature

pT181 Staphylococcus aureus 4.4 Tetracycline resistance

ColE1 Escherichia coli 6.6 Colicin production and immunity

pGKL2 Kluyveromyces lactisb 13.5 Killer plasmid

pAMβ1 Enterococcus faecalis 26.0 Erythromycin resistance

pSK41 Staphylococcus aureus 46.4 Multidrug resistance

pBM4000 Bacillus megaterium 53.0 rRNA operon

pI258 Staphylococcus aureus 28.0 Metal ion resistance

pSLT Salmonella enterica subsp. typhimurium 93.9 Virulence determinants

pMT1 Yersinia pestis 101.0 Virulence determinants

pADP-1 Pseudomonas sp. 108.8 Atrazine (herbicide) catabolism

pWW0 Pseudomonas putida 117.0 Aromatic hydrocarbon degradation

pBtoxis Bacillus thuringiensis subsp. israelensis 137.0 Mosquito larval toxicity

pX01 Bacillus anthracis 181.7 Exotoxin production

pSOL1 Clostridium acetobutylicum 192.0 Solvent production

pSymB Sinorhizobium meliloti 1683.3 Functions associated with plant symbiosis

Sources of genetic variation: point mutations

Sources of genetic variation: point mutations

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Sources of genetic variation: point mutations

Sources of genetic variation: DNA rearrangementsInsertion sequence (IS) elements:

1. Simplest type of transposable element found in bacterial chromosomes and plasmids.

2. Encode only genes for mobilization and insertion.

3. Range in size from 768 bp to 5 kb.

4. IS1 first identified in E. coli’s glactose operon is 768 bp long and is present with 4-19 copies in the E. coli chromosome.

5. Ends of all known IS elements show inverted terminal repeats (ITRs).

Sources of genetic variation: DNA rearrangements

Integration of IS element in chromosomal DNA.

Sources of genetic variation: DNA rearrangements

Sources of genetic variation: DNA rearrangements

Sources of genetic variation: frequency of occurrence

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Transmission of genetic variation: Luria-Delbruck test

Transmission of genetic variation: Luria-Delbruck test

Transmission of genetic variation: Luria-Delbruck test

Linear transmission of genetic variation

Horizontal transmission of genetic variation

Transmission of genetic variation: mechanisms

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Transmission of genetic variation: mechanisms

V. M. D'Costa et al. Science (2006)

Fig. 1. Antibiotic resistance profiling of 480 soil-derived bacterial isolates

Transmission of genetic variation: antibiotic resistance

Transmission of genetic variation: transformation

Transmission of genetic variation: transformation

• Gene transfer resulting from the uptake of DNA from a donor.• Factors affecting transformation

– DNA size and state• Sensitive to nucleases

– Competence of the recipient (Bacillus, Haemophilus, Neisseria, Streptococcus)

• Competence factor• Induced competence

Transmission of genetic variation: transformation

Transmission of genetic variation: transformation

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Transmission of genetic variation: transformation

Transmission of genetic variation: transduction

Transmission of genetic variation: transduction

Transmission of genetic variation: transduction

How did Zinder and Lederberg prove that the phenotype

was the result of transduction?

• presence of DNAase rules out transformation

• filter prevented contact so no conjugation

• reducing filter pore size to below size of phage inhibited

Transmission of genetic variation: transduction

QuickTime™ and aTIFF (LZW) decompressor

are needed to see this picture.

Transmission of genetic variation: transduction

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Transmission of genetic variation: transduction

Transmission of genetic variation: transduction

• There are two types of transduction:

– generalized transduction: A DNA fragment is transferred from one bacterium to another by a lytic bacteriophage that is now carrying donor bacterial DNA due to an error in maturation during the lytic life cycle.

– specialized transduction: A DNA fragment is transferred from one bacterium to another by a temperate bacteriophage that is now carrying donor bacterial DNA due to an error in spontaneous induction during the lysogenic life cycle

Transmission of genetic variation: transduction

1. A lytic bacteriophage adsorbs to a susceptible bacterium.

3. Occasionally, a bacteriophage head or capsid assembles around a fragment of donor bacterium's nucleoid or around a plasmid instead of a phage genome by mistake.

Transmission of genetic variation: generalized transduction

2. The bacteriophage genome enters the bacterium. The genome directs the bacterium's metabolic machinery to manufacture bacteriophage components and enzymes

4. The bacteriophages are released.

5. The bacteriophage carrying the donor bacterium's DNA adsorbs to a recipient bacterium

Transmission of genetic variation: generalized transduction

http://www.cat.cc.md.us/courses/bio141/lecguide/unit4/genetics/recombination/transduction/transduction.html

6. The bacteriophage inserts the donor bacterium's DNA it is carrying into the recipient bacterium .

7. The donor bacterium's DNA is exchanged for some of the recipient's DNA.

Transmission of genetic variation: generalized transduction

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1. A temperate bacteriophage adsorbs to a susceptible bacterium and injects its genome .

2. The bacteriophage inserts its genome into the bacterium's nucleoid to become a prophage.

Transmission of genetic variation: specialized transduction

3. Occasionally during spontaneous induction, a small piece of the donor bacterium's DNA is picked up as part of the phage's genome in place of some of the phage DNA which remains in the bacterium's nucleoid.

4. As the bacteriophage replicates, the segment of bacterial DNA replicates as part of the phage's genome. Every phage now carries that segment of bacterial DNA.

Transmission of genetic variation: specialized transduction

5. The bacteriophage adsorbs to a recipient bacterium and injects its genome.

6. The bacteriophage genome carrying the donor bacterial DNA inserts into the recipient bacterium's nucleoid.

http://www.cat.cc.md.us/courses/bio141/lecguide/unit4/genetics/recombination/transduction/spectran.html

Transmission of genetic variation: specialized transduction

Transmission of genetic variation: mechanisms

Transmission of genetic variation: conjugation